Chronic inflammatory disorders are a common medical problem, and their incidence increases with aging. The best recognized inflammatory disorder associated with aging is arthritis, a syndromic concept that embraces distinct disorders such as osteoarthritis (e.g. of the knees) and rheumatoid arthritis (e.g., the joints of the fingers). Apart from arthritis, there are other diseases that present with less obvious signs of inflammation. These include chronic obstructive pulmonary disease (COPD), which is a syndromic designation mainly comprised of emphysema, chronic bronchitis and asthma, a disorder of relapsing and remitting neural dysfunction, multiple sclerosis (MS), arteriosclerosis, psoriasis, a desquamating skin condition, and inflammatory bowel disorders (IBD) such as Crohn's disease and ulcerative colitis, both debilitating conditions affecting the upper and lower intestinal tract, respectively. Ulcerative colitis, moreover, is a risk factor for the evolution of colorectal cancer, a leading cause of terminal morbidity and mortality among industrial populations. Being hidden from view, these conditions are not typically known by lay people to be inflammatory in nature, yet pathologists have long demonstrated inflammatory processes in each of these. T-lymphocytes are important players in all of these diseases (Westermann, et al, Ann. Intern. Med. 2001, 135, 279). Many other disorders involve inflammatory phenomena, and they will not be reviewed exhaustively here.
The gross hallmarks of inflammation have been known since antiquity, when Roman physicians codified the four cardinal signs, the “four ors”, apparent by physical examination: dolor, rubor, calor and tumor. Dolor is pain, which is demonstrable while working with the patient. Rubor is the red appearance of the affected part, and this hyperemia is caused by the vascular dilatation and increased perfusion of inflamed tissue. This is the body's response to the demands of metabolically stimulated tissues. The dynamics of trafficking of cellular and subcellular components into the field of inflammation and out of it is dramatically increased relative to the basal state, and healing has significant costs in energy and nutritional requirements. The affected region is warmer to the touch than surrounding areas, and this is a reflection of the metabolic burden tissue injury entails. This increased metabolic demand and its consequent supply leads to the third sign, calor, or heat, as the tissue is warm to the touch. Finally, the fourth cardinal sign of inflammation is tumor, or swelling. The net effect of the inflammatory process is an increased amount of material in the affected part. The tissue is swollen with fluid because of increased vascular permeability, and the blood supply delivers a coordinated cast of cellular players such as leukocytes, and later, fibroblasts, first to deal with the cause of injury, and later to heal the injured part.
The traffic of cellular components into inflamed tissue, and their behavior there are highly regulated and orderly. It is not a haphazard process, and the details are becoming better understood. Part of the trafficking of inflamed tissue is a veritable symphony of pro-inflammatory signaling molecules. The cast of cellular players is controlled by factors such as hormones, cytokines and chemokines, and these afford opportunities for therapeutic intervention. The recruitment of specific leukocyte subpopulations, such as T-cells, is regulated by small proteins called chemokines.
Chemokines are a large family of small protein hormones that are among the various factors involved in inflammatory processes (Alexander et al, Br. J. Pharmacol., 2007, 150 (Suppl. 1) S25). The word chemokine is a contraction of the words chemotactic cytokine, and this belies their role. Chemokines attract leukocytes to areas of inflammation. Chemokines also have other effects, modulating the adhesion to extracellular proteins, proliferation and secretion of other factors (e.g., interferon-γ).
Chemokines regulate diverse processes (http://en.wikipedia.org/wiki/Chemokine). A major role of chemokines is to guide cellular migration. Cellular chemotaxis involves the movement of cells from an area with a low concentration of chemokine, to an area with a higher concentration of chemokine. Some chemokines seem to be more homeostatic in function: for example, they direct lymphocytes to the lymph nodes, where they participate in immune surveillance by interacting with antigen-presenting cells within the nodes. Some chemokines have effects on development, such as promoting or inhibiting the growth of new blood vessels—angiogenic and angiostatic effects. Such homeostatic chemokines seem to regulate the trafficking of cells in a day to day manner. Others are expressed in response to injury, and these inflammatory chemokines generally have chemoattractant and other actions on target leukocyte populations. These chemokines are typically induced by interleukin-1 or interferon-γ by various types of cells. Finally, many of the chemokines cause immune cells to release enzymatic and other factors.
Chemokines are small proteins of about 8 to 10 kDa in size stratified according to their protein sequence, and different cell populations respond to them based on the relevant receptors expressed on the cell surface (and Pease and Williams, Br. J. Pharmacol. 2006, 147, S212). To date, at least 47 chemokines are known, and they all have a basic “Greek key” protein folding motif consisting of three anti-parallel β-pleated sheets overlaid by a C-terminal α-helix. This protein fold depends upon conserved intra-chain disulfide bonds. The cysteines that form these bridges are the basis for chemokine nomenclature. Chemokines that have two consecutive cysteines near the amino terminus involved in disulfide bridges are called CC chemokines, and have the systematic name of CCL1 through CCl28. They bind to receptors that are similarly named, CCR1, CCR2, etc, although the numbering of the receptor is not the same as the binding chemokine, and multiple chemokines can bind to each receptor.
Chemokines with an amino acid between the first two cysteines of the amino terminus are called CxC chemokines. The systematic names for these hormones and their receptors are of the type CxCL1 through CxCL16 and the cognate receptors are CxCR1 through CxCR16. While other inter-cysteinyl spacings are plausible, the next class of chemokine known is Cx3CL1, called fractalkine, and it binds to the receptor Cx3CR1. Finally, there is at least one member of a class of chemokines with only one cysteine of the amino terminal region involved in a disulfide bond, called XCL1, lymphotactin, and its receptor XCR1. Taken altogether, there are 18 receptors known for the 47 known chemokines.
Importantly, splice variants have now become recognized, further subdividing the various chemokines and receptors that can exist. For CxCR3, there are two splice variants known: CxCR3A and CxCR3B. Chemokine receptors are 340-350 amino acids long, and all of them are G-protein coupled receptors (GPCRs). GPCRs are an important class of proteins targeted by various therapeutic agents including small molecule drugs. There is a long-felt need to modulate inflammation, and GPCRs are well-known to offer tractable targets for therapeutic intervention. Thus, there is much hope for the selective modulation of inflammatory processes by the use of modulators of chemokine receptors.
CxCR3A receptors are predominantly expressed on activated Th1 lymphocytes, but it is also present on NK (natural killer) cells, macrophages, DC (dendritic cells) and B lymphocytes. CxCR3A is known to be stimulated by three chemokines: CxCL9 (Monokine induced by interferon-γ, aka Mig), CxCL10, (Interferon-γ inducible 10 kDa protein, aka IP-10) and CxCL11 (Interferon-inducible T-cell α-chemoattractant, aka I-TAC). The observation of angiostatic effects of these chemokines presaged the possibility of a different receptor subtype, and indeed, a splice variant is discovered. CxCR3A signaling is mediated by a pertussis toxin sensitive G protein (Gαi), which causes a flux of calcium ions. The splice variant CxCR3B is found to be expressed on endothelial cells and mediates angiostatic effects of IP-10, Mig, I-TAC and Platelet Factor 4 (Lasagni et al, J. Exp. Med. 2003, 197, 1537). Platelet Factor 4, the first chemokine to be sequenced, has no effect on CxCR3A, and signaling through CxCR3B causes a rise of cAMP mediated by Gαs.
T lymphocytes have long been known to function as a command and control center of the immune system. Indeed, HIV causes AIDs by the selective destruction of T-cell populations. Given this central regulatory position, it is not surprising that of 18 chemokine receptors, 15 are expressed among the various subpopulations of T lymphocytes (Pease & Williams Br. J. Pharmacol. 2006, 147, S212).
Other pathogens are known to subvert the chemokine system to evade immune surveillance, suppress immune reactions and avoid elimination. Chemokine action is undermined by pathogens in at least four different ways: First, by production of chemokine mimics that act as receptor antagonists. Second, by producing chemokine mimics that act as inappropriate agonists. Third, by producing receptor mimics and fourth, by producing proteins that bind and neutralize chemokine activities (Chensue, Clin. Microbiol. Rev. 2001, 14, 821). CxCR3 and its ligands are among the factors that pathogens usurp. Thus, Nature has found ways to exploit chemokines to modulate functions of the immune system, and the subject of the present invention also exploits it.
Another important property of a control element of a physiological system is the presence of positive and negative regulatory inputs. These balancing points of integration are found among chemokines, too. Eotaxin/CCL11 and MCP-3/CCL7, while stimulatory for other chemokine receptors (CCR3 and CCR1, respectively), antagonize signaling by CxCR3A, and two other CCRs (CCR2 & CCR5). Importantly, the chemokines that stimulate CxCR3A (IP10/CxCL10, I-TAC/CxCL11 and Mig/CxCL9) antagonize signaling through CCR3 expressed on Eosinophils, Basophils and Mast cells (Alexander et al, Br. J. Pharmacol., 2007, 150 (Suppl. 1) S25). Thus, CxCR3A and its ligands are unique in how they are regulated and modulate the behavior of other chemokine signaling pathways. This receptor is at a highly interconnected node within the network of signaling molecules that coordinate inflammation. Indeed, the balance struck between CxCR3A versus CCR3 signaling determines the direction of the inflammatory response, polarizing it from parasite-fighting, allergic responses of the Th2 (CCR3) variety or towards the cell-mediated responses of Th1 (CxCR3A) type. This accounts for the “polarization” of the cellular component of inflammation (Loetscher, et al, J. Biol. Chem. 2001, 276, 2986). Thus, there is a long felt need to discover therapeutic agents that can intervene at this part of the immune response.
The highest level of expression of CxCR3A is seen in T cells, and it is one marker of Th1 lymphocytes (Annunziato, et al, Microbes and Infection, 1999, 1, 103; Lasagni et al, J. Exp. Med. 2003, 197, 1537). T cells are implicated in many diseases listed here in decreasing order of incidence: asthma, Grave's disease, Rheumatoid arthritis, atopic dermititis, Sjogren's syndrome, systemic lupus erythematosis, multiple sclerosis, ulcerative colitis, Type 1 diabetes mellitus, Crohn's disease, sarcoidosis, primary biliary cirrhosis, glomerulonephritis, myasthenia gravis, temporal arteritis, and allogeneic organ transplant rejection (Westermann, et al, Ann. Intern. Med. 2001, 135, 279). The therapeutic potential of CxCR3A modulators now will be illustrated by a few examples of experimental pharmacology.
CxCR3 and its ligands are associated with inflammatory bowel disease. Antibodies to IP10 decreased inflammation in murine models of colitis (Singh, et al, J. Immunol. 2003, 171, 1401). Knockout mice lacking IL-10 (IL-10−/−) spontaneously develop colitis that resembles Crohn's Disease. At about 12 weeks of age, these mice begin to lose weight, have chronic diarrhea and circulating levels of serum amyloid A, IL-6 and six other cytokines rise. Treatment with an IP10-neutralizing monoclonal antibody abrogated all of these effects. Histological examination also revealed that the antibody significantly reduced the extent of lymphocytic infiltration into the colonic mucosa.
In rheumatoid arthritis, CxCR3A agonist chemokines are elevated 100 fold in synovial fluid compared to samples from traumatic joint injury or osteoarthritis (Patel, et al, Clin. Immunol., 2001, 98, 39). They also are present in concentrations consistent with a gradient, high to low, from synovial fluid to plasma, and 94% of perivascular T cells express CxCR3A on their surface membranes, and this frequency is enriched over the 40% of T cells with the receptor on their plasma membranes in the blood stream. These findings are consistent with the theory that CxCR3A-binding chemokines are directing the recruitment of Th1 type T cells to the inflamed joints. Thus, for RA, the potential value of intervening at CxCR3A signaling is recognized by several authors (Houshmand & Zlotnik, Curr. Opinion Chem. Biol. 2003, 7, 457; Proudfoot, Nature Reviews Immunol., 2002, 2, 106).
A small molecule inhibitor of the CxCR3A system in arthritic models has been reported. Tak779, which antagonizes CCR5 and CxCR3A (both selectively expressed on Th1 cells—Hashmand & Zlotnick) inhibit pathology of CIA in mice (Yang, et al, Eur. J. Immunol. 2002, 32, 2124; and Gao, et al, J. Leukoc. Biol. 2003, 73, 273). The Collagen Induced Arthritis (CIA) model is a well-established acute arthritis model in mice with a course of 26 days. Mice are immunized with collagen, and within 13 days of the booster injection, nearly all animals have swollen red joints in their limbs. This classical picture of inflammation is ablated by treating CIA mice with TAK-779, a quarternary ammonium salt of a substituted benzocycloheptene originally developed as a drug to block HIV infection. TAK-779 also blocked the leukocytic infiltration of joints as determined by histologic evaluation. Although TAK-779 is originally developed as a CCR5 inhibitor to treat AIDS, it is found to be a CxCR3A blocker with similar potencies as measured in competitive radioligand binding, chemotaxis, and cellular adhesion assays. Two other chemokines expressed in the cell type studied by Gao et al had no effect on these parameters. Thus, TAK-779 is a dual inhibitor for both CCR5 and CxCR3A.
A small molecule compound, AMG-487, has been tested in a murine model of metastatic cancer (Walser, et al, Cancer Res., 2006, 66, 7701). Interestingly, tumor cells often aberrantly express chemokines or their receptors. Investigators believe that this may promote growth or affect tropism of metastatic disease. CxCR3A is expressed in breast cancer cell lines from humans and mice, they functionally couple to calcium responses and impart chemotactic activity to these cells responsive to CxCR3A-specific chemokines. In a mouse model of metastatic breast cancer, AMG-487 reduced the number of metastases by 60%. Interestingly, the compound had no direct effect on proliferation, emphasizing that metastatic tropism and proliferation are dissociable phenomena.
Finally, recent gene knockout experiments in mice predict utility for CxCR3 inhibitors in the treatment of arteriosclerosis (Veillard, et al, Circulation 2005, 112, 870). Mice lacking the ApoE gene rapidly develop atherosclerotic lesions in the aorta when given a high fat diet. When the gene for CxCR3 is knocked out in these mice, the extent of lipid deposits in the thoracoabdominal aorta is reduced from 7.9% of the area to 4.5%.
Thus, there is good reason to believe that modulators of CxCR3A will likely be useful for the treatment of patients with diverse diseases.
Assorted patent applications and issued patents discloses inhibitors of chemokines or CxCR3.
WO 2002/085861, WO 2003/101970, U.S. Pat. No. 7,067,662 B2, U.S. Pat. No. 6,124,319, WO 031070242 A1 and WO 2007/064553 A2 disclose respectively compounds of the following formulae:
as chemokine receptor inhibitors.
US 20070021611 A1, US 20070054919 A1, US 20070082913 A1, WO2008008453 A1, and WO2007/109238 A1 disclose respectively compounds of the following formulae:
as CxCR3 inhibitor. All these compounds have a (piperidin-4-ylpiperazin-1yl) aromatic moiety core structure.
WO 2007/002742 A1 discloses compounds of the following formula:
An illustrative compound of that series is as follows:
Cole, et al, J. Bioorg. Med. Chem. Lett. 16, 2006, 200-203, noted relative to the aforesaid series that the seven-membered homopiperazine ring is required for activity as the piperazinyl analogue exhibited not activity. Thus, the change of the homopiperazine ring to a piperazine ring is taught against.